Mu-Opioid Receptors on Nociceptors, Not Microglia, Drive Morphine Tolerance and Hyperalgesia in Mice

Researchers have long sought to minimize the side effects of opioids without sacrificing the unparalleled analgesia they offer. Now, Grégory Scherrer, Stanford University, US, and colleagues show that it’s possible to prevent tolerance and the paradoxical increase in pain sensitivity known as opioid-induced hyperalgesia (OIH) that can occur with chronic opioid use.

By genetically removing mu-opioid receptors (MORs) from peripheral nociceptive neurons, the researchers found that they could reduce both tolerance and OIH while maintaining analgesia in mice receiving daily doses of morphine.

“The main take-away from the paper is that you can dissociate the side effects of opioids from the main effect of analgesia,” said Howard Gutstein, University of Pittsburgh, US, who was not involved in the study. Gutstein co-authored with Stephanie Puig a News and Views commenting on the paper, as well as on another new paper showing that blocking spinal microglial pannexin-1 channels reduced morphine withdrawal in rodents without effects on analgesia (Puig and Gutstein, 2017; Burma et al., 2017).

What’s more, administration of morphine along with a peripherally restricted MOR antagonist also lessened tolerance and OIH without diminishing analgesia in models of perioperative and chronic pain. Since the antagonist, methylnaltrexone bromide (MNB), is an FDA-approved drug, there’s hope that the combined drug strategy could be effective and safe in patients who use opioids for pain relief.

“What’s exciting about these findings is that clinical trials could be set up pretty much immediately,” said Gutstein.

The study was published online January 16 in Nature Medicine.

A role for microglia?

Together, a handful of studies have made the case that opioids trigger tolerance and hyperalgesia by binding to MORs on dorsal horn microglia (see PRF related story). Based on his previous research and discussions with others in the field, however, Scherrer was not so sure that microglia actually express these receptors.

“He said, ‘Show me the data,’” according to Vivianne Tawfik, an anesthesiologist and pain medicine physician at Stanford University, and one of the lead authors of the current study. “So, I did a review of the literature. And I realized that the data on opioid receptors and glia weren’t very strong.”

Scherrer and Tawfik saw two potential issues. First, most previous studies had focused on cultured microglia rather than those in intact tissue. Second, the specificity of the immunolabeling generated by antibodies against opioid receptors, including for MOR, is not always established.

So, in the new work, Tawfik, along with fellow lead authors Gregory Corder, Dong Wang, Elizabeth Sypek, and colleagues, began by testing whether they could uncouple morphine-induced tolerance and hyperalgesia from microglial activation (visualized using the marker CD11b). When given injections of morphine twice daily, wild-type mice developed signs of these side effects as well as microglial activation, compared to mice that received saline. Global MOR knockout animals, however, only showed microglial activation. Chronic morphine, then, seemed to cause tolerance and hyperalgesia, but not microglial activation, through MORs.

As a possible explanation for these findings, the researchers failed to detect MOR messenger RNA or protein in microglia. But they found both in pain-signaling neurons (marked by calcitonin gene-related peptide) in the dorsal root ganglia.

“The authors argue that microglia do not express mu-opioid receptors, but their data do not argue that microglia are not involved [in tolerance and hyperalgesia],” wrote Yves De Koninck, Université Laval, Québec, Canada, in an email to PRF. De Koninck was not involved in the study.

“All the previous literature does suggest that there are signals released from neurons that act on microglia in response to opioids; it is clear that microglia are implicated,” said Corder. Such signals presumably bind to receptors other than MORs, recruiting microglia in the process (see PRF related story). But the results left the question of which MOR-expressing cells were responsible for tolerance and OIH.

A new culprit

So the researchers went on to test if morphine was instead acting on peripheral pain-sensing neurons, rather than microglia, to produce opioid side effects. Over 10 days, they gave daily subcutaneous injections of morphine to wild-type mice, as well as to mice with MORs genetically deleted from nociceptive neurons expressing the transient receptor potential vanilloid type 1 (TRPV1) ion channel.

In contrast to wild-type animals, mice lacking MORs in nociceptors showed reduced signs of both tolerance and hyperalgesia in response to morphine. Yet the two groups had similar levels of anti-nociception, showing comparable reflexive responses to heat and touch. “We also looked at motivational behaviors that occur after a painful stimulus is given, where the animal attends to the affected tissue and initiates escape behaviors,” said Corder. Here, too, the knockouts showed morphine anti-nociception similar to controls.

To test a more clinically relevant strategy, the researchers gave morphine along with MNB to wild-type mice. Importantly, MNB cannot cross the blood-brain barrier, restricting it to the periphery. When given daily for seven days, the combination of the two drugs, but not morphine alone, stopped tolerance and hyperalgesia from developing. On the other hand, analgesia, presumably mediated by central opioid action, was left intact. This strategy produced similar results in the tibia fracture and bone pinning model of perioperative pain, and the chronic constriction injury model of chronic pain.

This is not the first time researchers have teased apart the desired and undesired effects of opioids. In 2012, for instance, Gutstein and colleagues showed that they could prevent morphine tolerance, without affecting analgesia, in rats by blocking another receptor, the platelet-derived growth factor receptor-β, with the cancer drug imatinib (see PRF related story).

More recently, researchers have discovered so-called biased agonists for both the MOR and the kappa-opioid receptor. These agonists produce analgesia without affecting breathing, motor activity, or reward-related circuits, at least in rodents (see PRF related stories here and here).

But the combined drug strategy stands out because MNB is already used to treat opioid-induced constipation. As a result, “I don’t think MNB would need any other translational data” to move into clinical trials, said Tawfik. “I think our data put out there the possibility that we could improve these opioid side effects with this peripherally restricted drug.”

It’s a strategy that is sorely needed. Due to tolerance, patients who use opioids must often increase their dose to keep pain at bay. That escalation, however, exponentially increases the risk of mortality and morbidity, said Tawfik.

“This strategy is not encouraging the use of opioids. But for people who are taking them, is there a way we can decrease the risks?”

Matthew Soleiman is a science writer currently residing in Nashville, Tennessee. Follow him on Twitter @MatthewSoleiman.